Mri evaluation of heterogeneous tissue

ABSTRACT

The methods of the invention exploit the capabilities of manganese-enhanced MRI (MEMRI), which provides viability-specific biological contrast agent through the intracellular accumulation of Mn 2+ . A typical contrast agent utilizes non-chelated Mn 2+  in combination with Ca 2+ , e.g. as calcium gluconate. Active intracellular accumulation of Mn 2+  generates high signal from the viable cells in normal tissue, or normal regions of heterogeneous tissue; no signal from the non-viable cells; and intermediate signal from viable but injured cells. The intermediate signal defines a “gray zone” of potentially salvageable cells.

BACKGROUND OF THE INVENTION

Ischemic cardiomyopathy (ICM) is the primary etiology of advanced heartfailure (HF), the leading diagnosis of hospital admissions in the US.Clinical studies have confirmed that the high morbidity and mortality ofHF are associated with ventricular arrhythmias and LV remodeling in theperi-infarct region (PIR). Indeed, patients with a history of acute MIand LV dysfunction have a 6 month mortality >10%, one third of which isattributed to sudden cardiac death. The critical role of tissueheterogeneity in the PIR, independent of actual infarct size, has beenrecognized as an important substrate to trigger these ventriculararrhythmias. To this end, preclinical studies have confirmed that theheterogeneous PIR contains the critical isthmus for ventriculartachycardia and successful therapy requires ablation of the isthmus.

Studies have also demonstrated that revascularization of the ischemicPIR results in a lower incidence of ventricular arrhythmias and LVdilatation. While revascularization may mitigate ventricular arrhythmiasacutely, the Multicenter UnSustained Tachycardia Trial (MUSTT) notedthat following a reperfused MI, patients were increasingly likely tohave ventricular arrhythmias as their infarct matured. Therefore, anaccurate tissue characterization of the PIR is critical in determiningwhich patients will benefit from revascularization and/or medicaltherapy targeted at PIR.

In order to evaluate the role of the PIR, clinical studies have employedcardiac MRI (CMR) to assess PIR in patients with severe ICM, whichconfirmed that precise tissue characterization of the PIR predictsfuture cardiovascular events while traditional measures including scarpresence, LVEF, and LV volumes did not demonstrate such significance.The trials revealed the need for a more sensitive method to detectsalvageable myocardium within and around areas of cardiac injury toallow clinicians to guide therapy. Compositions and methods are providedfor as a means of distinguishing through imaging between necrosed cells,injured cells and fully viable cells in vivo, using manganese enhancedMRI.

U.S. Pat. No. 5,980,863 is directed to compositions for MRI imaging oftissue. Manganese-enhanced MRI (MEMRI) exploits the T1 shortening effectof Mn²⁺ to generate positive MRI contrast. Also see Chung et al. (2012)Magn Reson Med. 68(2):595-9; and Dash et al. (2011) Circ CardiovascImaging. 4(5):574-82.

SUMMARY OF THE INVENTION

Compositions and methods are provided for MRI analysis in vivo ofheterogeneous tissue, in which small numbers of viable cells may bepresent within tissue comprising non-viable cells. In some embodimentsthe heterogeneous tissue is adjacent to a region of necrosis, forexample an infarcted region.

The methods of the invention exploit the capabilities ofmanganese-enhanced MRI (MEMRI), which provides viability-specificbiological contrast agent through the intracellular accumulation ofMn²⁺. A contrast agent for use in the methods of the invention mayutilize non-chelated Mn²⁺ in combination with Ca²⁺, e.g. as calciumgluconate. Active intracellular accumulation of Mn²⁺ generates highsignal from the viable cells in normal tissue, or normal regions ofheterogeneous tissue; no signal from the non-viable cells; andintermediate signal from viable but injured cells. The intermediatesignal defines a “gray zone” of potentially salvageable cells.

In some embodiments of the invention, the MRI analysis of heterogeneoustissue is performed over time, where an individual is monitored for atleast two time points, and may be monitored over a series of timepoints. In some embodiments, therapeutic intervention, which may be apart of a clinical trial, is performed during the period of time betweenanalysis. therapeutic intervention may include, without limitation, celltherapies, e.g. introduction of regenerative cells to the tissue;biological therapies, such as gene therapy to introduce regenerativegenetic and/or proteins to the injured cells; contacting the cells withantibodies, cytokines, growth factors, and the like to modulate cellgrowth and/or regeneration; drug therapies, e.g. treating the patientwith a pharmaceutical agent to modulate the intermediate zone cells;revascularization and reperfusion treatments, e.g. stents, surgicalbypass and other revascularization modalities; environmental strategies;diet intervention; and other modes of therapy that may affect the growthand biological activity of the gray zone cells.

In some embodiments of the invention, a combination approach thediffering capabilities of MEMRI and delayed-enhanced MRI (DEMRI), whichdistributes primarily within the extracellular space to generate abright MRI signal in infarcted tissue; to enhance the identification andvisualization of the gray zone cells. The combined use of these methodsmay be referred to herein as dual contrast MRI. The MEMRI and DEMRIanalysis may be performed substantially concomitantly, or may beperformed sequentially, alternately, etc.

In some embodiments the tissue being imaged is damaged cardiac tissue.In some such embodiments the tissue being imaged is infarcted cardiactissue, e.g. in a patient following an episode of myocardial ischemia,and particularly in patients having or suspected of having myocardialinfarction, which may be transmural or non-transmural. In suchembodiments, the imaging identifies a bright signal from non-infarctedtissue, no signal from infarcted tissue, and an intermediate, gray zonesignal from viable cells within the peri-infarct region. The resultingpattern allows high-resolution, steady-state imaging of myocardialviability and injury. Cells providing an intermediate signal are ofparticular interest for analysis during therapeutic strategies, becausethese cells have potential for salvage and restoration of function, buthave also been identified as a source of ventricular arrhythmias.

In another embodiment, the cardiac tissue is atrial wall tissue, e.g.following atrial wall damage. Atrial wall damage can be included inassessment of myocardial infarction, but may be assessed for otherconditions, including without limitation: aneurysm; conditions relatingto atrial arrhythmia; left atrial (LA) wall injury after pulmonary veinantrum isolation (PVAI) in patients with atrial fibrillation (AF); leftatrial (LA) substrate remodeling (SRM) in patients with rheumatic mitralvalve disease and persistent atrial fibrillation (AF); followingcoronary artery bypass grafting (CABG); and the like. In suchembodiments, the imaging identifies a bright signal from normal tissue,no signal from non-viable tissue, and an intermediate, gray zone signalfrom viable cells within the damaged region. The resulting patternallows high-resolution, steady-state imaging of atrial wall viabilityand injury.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 The percentage Peri-Infarct Region (PIR) per total LV volume.

FIG. 2 The percentage Peri-Infarct Region (PIR) per DEMRI enhancedvolume.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Although preferred embodiments of the invention are described below, itshould be understood that the present disclosure is to be considered asan exemplification of the principles of this invention and is notintended to limit the invention to the embodiments illustrated.

The methods of the invention provide a means of distinguishing throughimaging between injured cells and fully viable cells in vivo, usingmanganese enhanced MRI. The application of a manganese imaging contrastagent to a living tissue of interest allows distinction based on theuptake of the contrast agent, which differs between dead or necroticcells, normal viable cells, and injured cells.

Heterogeneous tissue comprises cells that are heterogeneous with respectto viability, for example comprising dead, substantially normal, andinjured cells. The distribution and the level of injury is oftenvariable as well. It will be understood by one of skill in the art thatthere is a continuous range of physiologic activity, and that theguidelines provided herein are intended to encompass that naturalbiological variability.

The physiology of a cell may be characterized with reference to avariety of parameters that are associated with normal cell function,including without limitation utilization of substrates, for exampleglucose, O₂, and the like; with reference to pathway activity, includingwithout limitation glycolysis, citric acid cycle, oxidativephosphorylation, etc.; with reference to replicative ability ifapplicable, i.e. if a cell is normally dividing; with respect to thenormal function of the cell, for example contractility, synthesis ofhormones, excitation, and the like; with respect to the normal life spanof the normal cell; or any other suitable measure of viability.

The term viable cells refers to those cells having the cellular anatomyand/or physiologic activity of the cells normally resident in the tissueof interest. For example a potentially injured neural cell may becompared to a normal neural cell, a cardiomyocyte to a normalcardiomyocyte, and the like. Generally, a cell considered to be viablewill have at least 50% of the normal parameters of the cognate celltype, at least about 75% of the normal parameters, at least about 80% ofthe normal parameters, at least about 85% of the normal parameters, atleast about 90% of the normal parameters, at least about 95% of thenormal parameters, at least about 98%, at least about 99%, and may be100% or more.

Injured cells as defined herein have one or more reduced parametersassociated with normal cell function, as described above, and may have,for example, up to about 10%, up to about 20%, up to about 30%, up toabout 40%, up to about 50%, up to about 60%, up to about 70%, up toabout 80%, up to about 90%, up to about 95%, and less than about 100%.

There are many causes of cell injury, including without limitationhypoxia, ischemia, the presence of physical changes such as temperature,trauma, radiation; the presence of chemical agents such as toxins,drugs; etc.; the presence of infection; the presence of inflammatory orother immune responses; nutritional imbalance; genetic changes; and thelike.

The methods of the present invention may be benchmarked with respect tostructural characteristics of injured cells, which may include withoutlimitation cell swelling, detachment of ribosomes from granular e.r.,dissociation of polysomes into monosomes; pallor, hydropic change,vacuolar degeneration, plasma membrane blebbing, blunting, villousdistortion, myelin figures, mitochondrial swelling, rarefaction, nucleardisaggregation of granular and fibrillar elements.

Other examples of structural features useful in benchmarking cellinjuries include, without limitation, a scoring system based onultrastructure. In one example for analysis of cardiac tissue, eachidentified cardiomyocyte may be graded on whether it exhibited a highabundance (5), moderate abundance (4), low abundance (3), rare (2), orcomplete absence (1) of a feature. Conversely, for an unhealthy tissuefeature, each identified cardiomyocyte may be graded with 5 indicatingthat the nucleus displayed complete absence, 4 indicating rare, 3indicating low abundance, 2 indicating moderate abundance, and 1indicating high abundance of the unhealthy feature. For example, thecomplete absence of a healthy feature or the high abundance of anunhealthy feature yields a score of 1. Features include in the nucleus:notched/furrowed membrane; homogenous chromatin granules; chromatinaccumulated along nuclear membrane; chromatin clots within nucleus;dense chromatin; dark chromatin finely structured. Features inmitochondria include dense perinuclear accumulation; finefilaments/glycogen granules between nucleus and mitochondria; destroyedcristae; few mitochondria near nucleus; mitochondria isolated in niche.Features in myofibrils include myofibrils aligned in 1 row; t-tubulescontain basal lamina; myofibrils are contracted; z-line disruption;lipid droplets between ruptured myofibrils.

The difference in staining between viable and injured cells provides auseful guide for therapy, in that tissue containing injured but not deador infarcted cells may be amenable to treatment. Staining densities maybe compiled into a signature pattern that is useful for prognosis andfor theranostic purposes.

Generally with respect to an infarct, or other region of scarring orregion of necrosis, a remote tissue is considered to be viable orsubstantially normal, the infarct or other damaged region can containnon-viable cells, and the “border” are cells that are injured but may besalvaged with therapy. The difference in intensity of staining is fromabout 1.25-fold, about 1.5-fold, about 1.75-fold, about 2-fold, about2.25 fold, about 2.5 fold or greater decreased between the remote(viable) cells and injured cells. The difference in intensity ofstaining is from about 3-fold, about 3.5-fold, about 4-fold, about4.5-fold, about 5-fold or greater decreased between the remote (viable)cells and dead cells in the infarct. The difference in intensity ofstaining is from about 2.5-fold, about 2.75-fold, about 3-fold, about3.25-fold, about 3.5-fold, about 3.75-fold, about 4-fold, about4.25-fold or greater increased between the injured (or gray zone) cellsand dead cells in the non-viable region.

The signature pattern may be generated from imaging data as describedabove. The readout may be a mean, average, median or the variance orother statistically or mathematically-derived value associated with themeasurement. Following obtainment of the signature pattern from thesample being assayed, the signature pattern can be compared with areference or control profile to make a prognosis regarding the phenotypeof the tissue from which the sample was obtained/derived. Typically acomparison is made with a sample or set of samples from an unaffected,normal source, from a known diagnosis, etc. An algorithm that compilesthe imaging results will discriminate between individuals in differentclassifications with respect to prognosis for response to therapy.

An analytic classification process may use any one of a variety ofstatistical analytic methods to manipulate the quantitative data andprovide for classification of the sample. Examples of useful methodsinclude linear discriminant analysis, recursive feature elimination, aprediction analysis of microarray, a logistic regression, a CARTalgorithm, a FlexTree algorithm, a LART algorithm, a random forestalgorithm, a MART algorithm, machine learning algorithms; etc.Classification can be made according to predictive modeling methods thatset a threshold for determining the probability that a sample belongs toa given class. The probability preferably is at least 50%, or at least60% or at least 70% or at least 80% or higher. Classifications also maybe made by determining whether a comparison between an obtained datasetand a reference dataset yields a statistically significant difference.If so, then the sample from which the dataset was obtained is classifiedas not belonging to the reference dataset class. Conversely, if such acomparison is not statistically significantly different from thereference dataset, then the sample from which the dataset was obtainedis classified as belonging to the reference dataset class.

The predictive ability of a model may be evaluated according to itsability to provide a quality metric, e.g. AUC or accuracy, of aparticular value, or range of values. In some embodiments, a desiredquality threshold is a predictive model that will classify a sample withan accuracy of at least about 0.7, at least about 0.75, at least about0.8, at least about 0.85, at least about 0.9, at least about 0.95, orhigher. As an alternative measure, a desired quality threshold may referto a predictive model that will classify a sample with an AUC (areaunder the curve) of at least about 0.7, at least about 0.75, at leastabout 0.8, at least about 0.85, at least about 0.9, or higher. As isknown in the art, the relative sensitivity and specificity of apredictive model can be “tuned” to favor either the selectivity metricor the sensitivity metric, where the two metrics have an inverserelationship. The limits in a model as described above can be adjustedto provide a selected sensitivity or specificity level, depending on theparticular requirements of the test being performed. One or both ofsensitivity and specificity may be at least about at least about 0.7, atleast about 0.75, at least about 0.8, at least about 0.85, at leastabout 0.9, or higher.

Cardiac Indication and Uses

In some embodiments of the invention, the tissue being analyzed is hearttissue. The heart needs to be supplied with a sufficient quantity ofoxygen to prevent underperfusion. When reduced perfusion pressure distalto stenoses is not compensated by autoregulatory dilation of theresistance vessels, ischemia, meaning a lack of blood supply and oxygen,occurs. Because the zone least supplied generally is the farthest out,ischemia generally appears in areas farthest away from the blood supply.

After total or near-total occlusion of a coronary artery, myocardialperfusion occurs by way of collaterals, meaning vascular channels thatinterconnect epicardial arteries. Collateral channels may form acutelyor may preexist in an under-developed state before the appearance ofcoronary artery disease. Preexisting collaterals are thin-walledstructures ranging in diameter from 20 μm to 200 μm, with a variabledensity among different species. Preexisting collaterals normally areclosed and nonfunctional, because no pressure gradient exists to driveflow between the arteries they connect. After coronary occlusion, thedistal pressure drops precipitously and pre-existing collaterals openvirtually instantly.

The term “myocardial ischemia” refers to a decrease in blood supply andoxygen to the cells of the myocardium. The development of myocardialischemia has been attributed to two mechanisms: (1) increased myocardialoxygen demand, and (2) decreased myocardial perfusion and oxygendelivery. Myocardial ischemia generally appears first and is moreextensive in the subendocardial region, since these deeper myocardiallayers are farthest from the blood supply, with greater need for oxygen.

Chronic Myocardial lschemia. The term “chronic myocardial ischemia(CMI)” as used herein refers to a prolonged subacute or chronic state ofmyocardial ischemia due to narrowing of a coronary blood vessel in whichthe myocardium “hibernates”, meaning that the myocardium downregulatesor reduces its contractility, and hence its myocardial oxygen demand, tomatch reduced perfusion, thereby preserving cellular viability andevading apoptosis. The underlying mechanism by which the myocardium doesso is poorly understood. This hibernating myocardium is capable ofreturning to normal or near-normal function on restoration of anadequate blood supply. Once coronary blood flow has been restored tonormal or near normal and ischemia is resolved, however, the hibernatingmyocardium still does not contract. This flow-function mismatchresulting in a slow return of cardiac function after resolution ofischemia has been called stunning. The length of time for function toreturn is quite variable, ranging from days to months, and is dependenton a number of parameters, including the duration of the originalischemic insult, the severity of ischemia during the original insult,and the adequacy of the return of the arterial flow.

Acute Myocardial Infarction (AMI). Another type of insult occurs duringAMI. AMI is an abrupt change in the lumen of a coronary blood vesselwhich results in ischemic infarction, meaning that it continues untilheart muscle dies. On gross inspection, myocardial infarction can bedivided into two major types: transmural infarcts, in which themyocardial necrosis involves the full or nearly full thickness of theventricular wall, and subendocardial (nontransmural) infarcts, in whichthe myocardial necrosis involves the subendocardium, the intramuralmyocardium, or both, without extending all the way through theventricular wall to the epicardium. There often is total occlusion ofthe vessel with ST segment elevation because of thrombus formationwithin the lumen as a result of plaque rupture. The prolonged ischemicinsult results in apoptotic and necrotic cardiomyocyte cell death.Necrosis compromises the integrity of the sarcolemmal membrane andintracellular macromolecules such that serum cardiac markers, such ascardiac-specific troponins and enzymes, such as serum creatine kinase(CK), are released. In addition, the patient may have electrocardiogram(ECG) changes because of full thickness damage to the muscle.

Acute myocardial infarction remains common with a reported annualincidence of more than one million cases in the United States alone.Preclinical and clinical data demonstrate that following a myocardialinfarction, the acute loss of myocardial muscle cells and theaccompanying peri-infarct zone hypoperfusion result in a cascade ofevents causing an immediate diminution of cardiac function, with thepotential for long term persistence. The extent of myocardial cell lossis dependent on the duration of coronary artery occlusion, existingcollateral coronary circulation and the condition of the cardiacmicrovasculature. Because myocardial cells have virtually no ability toregenerate, myocardial infarction leads to permanent cardiac dysfunctiondue to contractile-muscle cell loss and replacement with nonfunctioningfibrotic scarring. Moreover, compensatory hypertrophy of viable cardiacmuscle leads to microvascular insufficiency that results in furtherdemise in cardiac function by causing myocardial muscle hibernation andapoptosis of hypertrophied myocytes in the peri-infarct zone.

Among survivors of myocardial infarction, residual cardiac function isinfluenced by the extent of ventricular remodeling. The term ventricularremodeling refers to alteration in ventricular architecture, withassociated increased volume and altered chamber configuration, driven ona histologic level by a combination of pathologic myocyte hypertrophy,myocyte apoptosis, myofibroblast proliferation, and interstitialfibrosis. Although originally described after myocardial infarction(MI), ventricular remodeling develops in response to a variety of formsof myocardial injury and increased wall stress.

Alterations in ventricular topography (meaning the shape, configuration,or morphology of a ventricle) occur in both infarcted and healthycardiac tissue after myocardial infarction. Ventricular dilatation(meaning a stretching, enlarging or spreading out of the ventricle)causes a decrease in global cardiac function and is affected by theinfarct size, infarct healing and ventricular wall stresses. Recentefforts to minimize remodeling have been successful by limiting infarctsize through rapid reperfusion (meaning restoration of blood flow) usingthromobolytic agents and mechanical interventions, including, but notlimited to, placement of a stent, along with reducing ventricular wallstresses by judicious use of pre-load therapies and proper after-loadmanagement. Regardless of these interventions, a substantial percentageof patients experience clinically relevant and long-term cardiacdysfunction after myocardial infarction. Despite revascularization ofthe infarct related artery circulation and appropriate medicalmanagement to minimize ventricular wall stresses, a significantpercentage of these patients experience ventricular remodeling,permanent cardiac dysfunction, and progressive deterioration of cardiacfunction, and consequently remain at an increased lifetime risk ofexperiencing adverse cardiac events, including death.

At the cellular level, immediately following a myocardial infarction,transient generalized cardiac dysfunction uniformly occurs. In thesetting of a brief (i.e., lasting three minutes to five minutes)coronary artery occlusion, energy metabolism is impaired, leading todemonstrable cardiac muscle dysfunction that can persist for up to 48hours despite immediate reperfusion. Coronary artery occlusion of moresignificant duration, i.e., lasting more than five minutes, leads tomyocardial ischemia and is associated with a significant inflammatoryresponse that begins immediately after reperfusion and can last for upto several weeks.

The Peri-Infarct Border Zone. The zone of dysfunctional myocardiumproduced by coronary artery occlusion extends beyond the infarct regionto include a variable boundary of adjacent normal appearing tissue. Thisischemic, but viable, peri-infarct zone of tissue separates the centralzone of progressive necrosis from surrounding normal myocardium. Theperi-infarct zone does not correlate with enzymatic parameters ofinfarct size and is substantially larger in small infarcts.

It is known that after an AMI, transient ischemia occurs in the borderzones and that percutaneous coronary interventions, which open up theinfarct-related artery, can adversely affect the health of theperi-infarct border zones. It has been suggested that intermediatelevels of mean blood flow can exist as the result of admixture ofpeninsulas of ischemic tissue intermingled with regions of normallyperfused myocardium at the border of an infarct. Progressive dysfunctionof this peri-infarct myocardium over time may contribute to thetransition from compensated remodeling to progressive heart failureafter an AMI.

Atrial fibrillation or other types of atrial arrhythmia. (AF or A-fib)is an abnormal heart rhythm characterized by rapid and irregularbeating. Heart-related risk factors include heart failure, coronaryartery disease, cardiomyopathy, and congenital heart disease. Valvularheart disease often occurs as a result of rheumatic fever. Lung-relatedrisk factors include COPD, obesity, and sleep apnea. AF is often treatedwith medications to achieve rate control or rhythm control.

AF is usually accompanied by symptoms related to a rapid heart rate.Rapid and irregular heart rates may be perceived as palpitations orexercise intolerance and occasionally may produce anginal chest pain (ifthe high heart rate causes ischemia). Other possible symptoms includecongestive symptoms such as shortness of breath or swelling. Thearrhythmia is sometimes only identified with the onset of a stroke or atransient ischemic attack (TIA). Some genetic conditions are associatedwith AF.

The primary pathologic change seen in atrial fibrillation is theprogressive fibrosis of the atria. This fibrosis is due primarily toatrial dilation; however, genetic causes and inflammation may be factorsin some individuals. Dilation of the atria can be due to almost anystructural abnormality of the heart that can cause a rise in thepressure within the heart. This includes valvular heart disease (such asmitral stenosis, mitral regurgitation, and tricuspid regurgitation),hypertension, and congestive heart failure. Any inflammatory state thataffects the heart can cause fibrosis of the atria. This is typically dueto sarcoidosis but may also be due to autoimmune disorders that createautoantibodies against myosin heavy chains. Mutation of the lamin ACgene is also associated with fibrosis of the atria that can lead toatrial fibrillation.

Once dilation of the atria has occurred, this begins a chain of eventsthat leads to the activation of the renin aldosterone angiotensin system(RAAS) and subsequent increase in matrix metalloproteinases anddisintegrin, which leads to atrial remodeling and fibrosis, with loss ofatrial muscle mass. Fibrosis is not limited to the muscle mass of theatria and may occur in the sinus node (SA node) and atrioventricularnode (AV node), correlating with sick sinus syndrome.

Radiofrequency ablation to achieve PVAI is a promising approach tocuring AF. Controlled lesion delivery and scar formation within the LAare indicators of procedural success, but the assessment of thesefactors has been limited to invasive methods. Noninvasive evaluation ofLA wall injury to assess permanent tissue injury can be an importantstep in improving procedural success. The Maze procedure is an effectiveinvasive surgical treatment that is designed to create electrical blocksor barriers in the atria of the heart, forcing electrical impulses thatstimulate the heartbeat to travel down to the ventricles.

The term “cardiac biomarkers” refers to enzymes, proteins and hormonesassociated with heart function, damage or failure that are used fordiagnostic and prognostic purposes. Different cardiac biomarkers havedifferent times that their levels rise, peak, and fall within the body,allowing them to be used, not only to track the progress of a heartattack, but to estimate when it began and to monitor for recurrence.Some of the tests are specific for the heart while others also areelevated with skeletal muscle damage. Current cardiac biomarkersinclude, but are not limited to CK (creatine phosphokinase or creatinekinase) and CK-MB (creatine kinase-myoglobin levels (to help distinguishbetween skeletal and heart muscle)), troponin (blood levels of troponinI or T will remain high for 1-2 weeks after a heart attack; troponingenerally is not affected by damage to other muscles), myoglobin (todetermine whether muscle, particularly heart muscle, has been injured),and BNP (brain natriuretic peptide) or NT-proBNP (N-terminal prohormonebrain natriuretic peptide (to help diagnose heart failure and grade theseverity of that heart failure).

The term “cardiac catheterization” refers to a procedure in which acatheter is passed through an artery to the heart, and into a coronaryartery. This procedure produces angiograms (i.e., x-ray images) of thecoronary arteries and the left ventricle, the heart's main pumpingchamber, which can be used to measure pressures in the pulmonary artery,and to monitor heart function.

The term “disease” or “disorder”, as used herein, refers to animpairment of health or a condition of abnormal functioning. The term“syndrome,” as used herein, refers to a pattern of symptoms indicativeof some disease or condition. The term “condition”, as used herein,refers to a variety of health states and is meant to include disordersor diseases caused by any underlying mechanism or disorder, injury, andthe promotion of healthy tissues and organs.

Methods of Analysis

An individual suspected of having an infarct or other region of tissuenecrosis, including without limitation fibrosis, scarring etc.associated with atrial fibrillation and treatment thereof, is analyzedby MEMRI for the presence of intermediate, or gray zone cells in regionssurrounding the necrotic or fibrotic tissue. Any tissue may be analyzed.In certain such embodiments the infarct is a myocardial infarct, and theindividual has been diagnosed with MI, or suspected of having MI. Inother embodiments the tissue is atrial wall tissue.

The Mn contrast agent may be any agent that provides Mn ions safely tothe patient. In some embodiments, the MEMRI contrast agent containsnon-chelated Mn²⁺ (12%) with calcium gluconate (10%). Although notrequired, toxicity modifiers could be used with the contrast agent. Thecontrast agent may be administered intravenously as a bolus or as aninfusion over a period of time. Commonly, though not necessarily, theinfusion will be over a period of 1 minute to 30 minutes. Larger dosesimprove the imaging of organs, such as the heart, that take up manganeseless efficiently than does liver. One may slow the rate ofadministration to increase the duration of signal intensity enhancementof blood without increasing total dose.

In a preferred embodiment, Mn gluconate/Ca gluconate (1:8), isparenterally administered over periods ranging from 10 seconds to 20minutes. Dosing is related to target organ of interest and may rangefrom from 1 μmol/kg body weight to 100 μmol/kg body weight of a sourceof Mn++ ion together with from 2 μmol/kg body weight to 1400 μmol/kgbody weight of a source of Ca++ ions. Preferably the source of manganeseis administered at 2 μmol/kg body weight to 30 μmol/kg body weight andthe source of calcium is administered at 4 μmol/kg body weight to 400μmol/kg body weight. Most preferably the source of manganese isadministered at 3 μmol/kg body weight to 15 μmol/kg body weight and thesource of calcium is administered at 6 μmol/kg body weight to 200μmol/kg body weight. MRI is performed from during or immediately postdosing to 24 hours post dosing (vascular indications excepted). The rateof administration may be varied to further improve the cardiovasculartolerability of the contrast agent without an adverse effect on imagequality, to increase the duration of the vascular phase of the agent, orto increase the dose without reducing the therapeutic index of the agentin order to enable imaging of target organs that accumulate manganeseless efficiently than does liver.

Following administration of the contrast agent, MRI imaging isperformed. MEMRI parameters, including T₁ and T₂ values, are measured.The data are analyzed to extract T₁ and T₂ values through nonlinearleast-square fits to the inversion recovery and spin-echo decay curves,respectively. These unique in vivo properties allow the infarcted tissueto take up negligible amount, peri-infarct regions reduced amount, andnormal tissue greatest amount for MEMRI enhancement. These propertiesenable precise determination of the baseline cardiac injury andsubsequent restoration by delineating the direct changes in the regionalviability at a cellular level.

By quantifying T1 values for each voxel in the myocardium, a parametricmap can be generated representing the T1 relaxation times of any regionof the heart without the need to compare it to a normal referencestandard before or after the use of a contrast agent. Alternativeimplementation of T1-mapping useds variable sampling of the k-space intime (VAST), acquiring images in three to four breath-holds andcorrelating that data to invasive biopsy. Other sequences have been usedfor quantification of T1 as well using inversion recovery TrueFISP ormultishot saturation recovery images. The most widely used T1-mappingsequence is based on the Modified LookLocker Inversion-recovery (MOLLI)technique. It consists of a single shot image with acquisitions overdifferent inversion time readouts allowing for magnetization recovery ofa few seconds after 3 to 5 readouts.

In some embodiments, dual contrast MEMRI-DEMRI analysis is performed.The novel dual contrast MEMRI-DEMRI can point to the viablecardiomyocytes within the PIR of transmural delayed enhancement. Thediscrepancy lies within the PIR that are positive for both MEMRI(viable) and DEMRI (non-viable) signal. The PIRs also display lowersignal to noise ratio (SNR) by MEMRI than remote zones and lower SNR byDEMRI than core infarct zones, reflecting the heterogeneity of PIR withsignificant population of viable cardiomyocytes with intact Ca²⁺-channelfunction (MEMRI positive) in the PIR of the adjacent necrotic tissue(DEMRI positive).

The information thus obtained regarding the status of intermediate zonetissue is particularly relevant for analysis of patients undergoingtherapy, and to assess the efficacy of clinical trials and treatmentmodalities in restoring function of the damaged tissue.

In some preferred embodiments, the methods of the invention are used indetermining the efficacy of a therapy for treatment of a cardiovasculardisease, either at an individual level, or in the analysis of a group ofpatients, e.g. in a clinical trial format. Such embodiments typicallyinvolve the comparison of two time points for a patient or group ofpatients. The patient status is expected to differ between the two timepoints as the result of a therapeutic agent, therapeutic regimen, ordisease challenge to a patient undergoing treatment.

The terms “therapeutically effective”, “infarct area-improving amount”,“perfusion improving amount” or “pharmaceutically effective amount”refer to a therapeutic dose or regimen that result in a therapeutic orbeneficial effect following its administration to a subject. The infarctarea-improving, infarct area-improving, perfusion-improving,therapeutic, or pharmaceutical effect may be curing, minimizing,preventing or ameliorating a disease or disorder, or may have any otherinfarct area-improving, infarct area-improving, perfusion-improving,therapeutic, or pharmaceutical beneficial effect. The effective amountof the composition may vary with the age and physical condition of thebiological subject being treated, the severity of the condition, theduration of the treatment, the nature of concurrent therapy, the timingof the infusion, the specific compound, composition or other activeingredient employed, the particular carrier utilized, and like factors.

Examples of formats for such embodiments may include, withoutlimitation, MEMRI or MEMRI-DEMRI analysis at two or more time points,where a first time point is a diagnosed but untreated patient; and asecond or additional time point(s) is a patient treated with a candidatetherapeutic agent or regimen. An additional time point may include apatient treated with a candidate therapeutic agent or regimen, andchallenged for the disease, for example a cardiac stress test.

In such clinical trial formats, each set of time points may correspondto a single patient, to a patient group, e.g. a cohort group, or to amixture of individual and group data. Additional control data may alsobe included in such clinical trial formats, e.g. a placebo group, adisease-free group, and the like, as are known in the art. Formats ofinterest include crossover studies, randomized, double-blind,placebo-controlled, parallel group trial is also capable of testing drugefficacy, and the like. See, for example, Clinical Trials: AMethodologic Perspective Second Edition, S. Piantadosi,Wiley-Interscience; 2005, ISBN-13: 978-0471727811; and Design andAnalysis of Clinical Trials: Concepts and Methodologies, S. Chow and J.Liu, Wiley-Interscience; 2003; ISBN-13: 978-0471249856, each hereinspecifically incorporated by reference.

In one embodiment, a blinded crossover clinical trial format isutilized. A patient alternates for a set period of time, e.g. one week,two weeks, three weeks, or from around about 7-14 days, or around about10 days, between a test drug and placebo, with a 4-8 week washoutperiod. In another embodiment a randomized, double-blind,placebo-controlled, parallel group trial is used to test drug efficacy.

In all such methods, the MEMRI or MEMRI-DEMRI analysis is performed atmultiple time points, with a setting and administration protocol thatpermits the detection of intermediate zone cells in the PIR.

Databases of Analyses

Also provided are databases of MRI analyses. Such databases willtypically comprise analysis profiles of various individuals following aclinical protocol of interest etc., where such profiles are furtherdescribed below.

The profiles and databases thereof may be provided in a variety of mediato facilitate their use. “Media” refers to a manufacture that containsthe expression profile information of the present invention. Thedatabases of the present invention can be recorded on computer readablemedia, e.g. any medium that can be read and accessed directly by acomputer. Such media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as CD-ROM; electrical storage mediasuch as RAM and ROM; and hybrids of these categories such asmagnetic/optical storage media. One of skill in the art can readilyappreciate how any of the presently known computer readable mediums canbe used to create a manufacture comprising a recording of the presentdatabase information. “Recorded” refers to a process for storinginformation on computer readable medium, using any such methods as knownin the art. Any convenient data storage structure may be chosen, basedon the means used to access the stored information. A variety of dataprocessor programs and formats can be used for storage, e.g. wordprocessing text file, database format, etc.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the informationof the present invention. The minimum hardware of the computer-basedsystems of the present invention comprises a central processing unit(CPU), input means, output means, and data storage means. A skilledartisan can readily appreciate that any one of the currently availablecomputer-based system are suitable for use in the present invention. Thedata storage means may comprise any manufacture comprising a recordingof the present information as described above, or a memory access meansthat can access such a manufacture.

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. Such presentation provides a skilled artisanwith a ranking of similarities and identifies the degree of similaritycontained in the test expression profile.

Additional Analysis

In combination with the methods of the invention, other methods ofcardiovascular analysis may be used, including various well-knownimaging techniques such as scintigraphy, myocardial perfusion imaging,gated cardiac blood-pool imaging, first-pass ventriculography,right-to-left shunt detection, positron emission tomography, singlephoton emission computed tomography, harmonic phase magnetic resonanceimaging, echocardiography, and myocardial perfusion reserve imaging.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges also is encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the described invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

As used herein and in the appended claims, the singular forms “a”,“and”, and “the” include plural referents unless the context clearlydictates otherwise. All technical and scientific terms used herein havethe same meaning.

Each of the references cited herein is incorporated herein by referencein its entirety. The publications discussed herein are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that thedescribed invention is not entitled to antedate such publication byvirtue of prior invention. Further, the dates of publication providedmay be different from the actual publication dates which may need to beconfirmed independently.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXPERIMENTAL EXAMPLE 1

Our first clinical study demonstrated that the peri-infarct (PIR)ischemia detected by cardiac MRI (CMR) predicted future cardiovascularevents (CVE) in this patient population. Our second clinical studyreported that the scar non-transmurality and volume/percentage of scarpredicted future CVE. Others have also shown that PIR lead to gradualdeterioration of cardiac function and pathological left ventricular (LV)remodeling with ongoing apoptosis and collagen deposition. Our thirdclinical study confirmed that tissue heterogeneity in the non-transmuralPIR predicted CVE. Other studies have also documented that the clinicalimportance of tissue heterogeneity in the PIR is independent of theactual infarct size. These studies confirmed that precise tissuecharacterization of the PIR was a significant predictor of future CVEwhile other traditional measurements, including scar presence, LVejection fraction (LVEF), and LV volumes did not demonstrate anysignificant relationship with the incidence of CVE. Additionally, thetissue heterogeneity in the PIR of ICM has been recognized as animportant substrate to trigger ventricular arrhythmias and inducepathological LV remodeling. Studies have confirmed that theheterogeneous PIR contains the critical isthmus for ventriculartachycardia and ablation therapy of PIR eliminates thearrhythmogenicity. Indeed, an effective diagnosis of PIR represents acritical unmet need in cardiovascular medicine.

Limitations of delayed-enhanced MRI (DEMRI). The current gold standardfor myocardial viability is delayed-enhanced MRI (DEMRI), which predictsfunctional recovery and improved survival in ICM patients afterrevascularization. This technique exploits the T1-shortening effect ofgadolinium (Gd), which distributes primarily within the extracellularspace to generate a bright MRI signal in acutely or chronicallyinfarcted myocardium. However, DEMRI does not provide direct cellviability information due to its nonspecific distribution properties.Despite the predictive capability of DEMRI, several groups have reportedthat DEMRI decreases significantly over time. Others have reported thatDEMRI may overestimate areas of nonviable infarct by as much as 15% withthe majority of the discrepancy lying within the heterogeneous PIR.Indeed, DEMRI may also be positive in regions of myocardial edema andinflammation, which may cause transient, reversible cardiac injurypatterns. To date, there is limited ability to detect viable and,potentially, salvageable cardiomyocytes within the PIR of DEMRIterritory. No established imaging strategy identifies these salvageableareas, which could have a meaningful survival impact for the patients.An alternative approach utilizing additional contrast agents tocomplement Gd may be necessary .

An alternative approach to evaluate myocardial viability ismanganese-enhanced MRI (MEMRI). A biologically active contrast agent,manganese (Mn²⁺), specific to the viable cardiomyocytes will complementthe anatomical DEMRI data of the injured myocardium. Mn²⁺ is a divalentcation that enters cells via voltage-gated calcium channels. MEMRIexploits the T1 shortening effect of Mn²⁺ and limits the uptake toviable, metabolically active cells. The intracellular accumulation ofMn²⁺ increases positive MRI signal intensity due to a T1-shorteningeffect, targeting the viable cardiomyocytes robustly. Mn²⁺ is anaturally occurring essential micronutrient and an antioxidant excretedvia the hepatobiliary system. Recent MnDPPP chelation and Ca²⁺supplementation have enabled clinical applications by eliminatingcardiac and neurologic toxicity. MEMRI improves the characterization ofthe injured myocardium and allows close correlation with histopathology.The resulting pattern of intracellular enhancement allows direct,high-resolution, steady-state imaging of myocardial viability. Itsuptake is dependent upon the metabolic function of the viable cells.

Although Mn²⁺ was recognized as the first MRI contrast agent, toxicityhindered widespread use. However, the use of non-chelated Mn²⁺ (12%)with Ca²⁺ supplementation (calcium gluconate 10%) in EVP1001 has enabledclinical application by eliminating toxicity. With 5 unpaired electrons,Mn²⁺ is among the most effective of all potential metal ion-based MRcontrast agents. Additionally, Mn²⁺ is a naturally occurring essentialmicronutrient, a natural antioxidant, and excreted via hepatobiliarysystem. Used widely in neuronal imaging, the uptake of Mn²⁺ into viablemyocardial cells has also been well-documented. Recent MEMRI studieshave demonstrated improved tissue characterization of the infarctedmyocardium through direct visualization of the viable myocardium andfound close correlation with histopathology of the infarct volumes.

Active intracellular accumulation of Mn²⁺ generates high signal from theviable cardiac cells in normal region, no signal from the non-viablecells in the intra-infarct region (IIR), and intermediate signal fromthe viable but injured cells in the peri-infarct region (PIR). Theresulting pattern of intracellular enhancement allows high-resolution,steady-state imaging of myocardial viability and injury.

In order to delineate the PIR, dual contrast MEMRI and delayed enhancedMRI (MEMRI-DEMRI) strategy has been developed to detect the viablemyocardium within the PIR where there are both positive DEMRI scar(non-viable) and positive MEMRI signal (viable). This heterogeneityindicates that a significant population of cardiomyocytes may be alivewith intact Ca²⁺-channel function (MEMRI positive) in this regiondespite large amounts of surrounding necrotic tissue (DEMRI positive).Indeed, our tissue electron microscopy analysis of these overlappingzones of MEMRI and DEMRI signal demonstrate morphologically andultrastructurally intact cardiomyocytes within the DEMRI-positive zones,corroborating the presence of viable and salvageable cardiomyocytes inthe PIR. These injured but viable cardiomyocytes may trigger ventriculararrhythmia and/or LV remodeling in HF. A definitive study to evaluatehow cell therapy alters these pockets of viable tissue directly willimpact the clinical strategies for this critical unmet clinical need incardiovascular medicine.

Mechanism and Safety Profile of MEMRI Contrast Agent. The ability ofMEMRI to detect the viable myocardium directly allows precise evaluationof the progressive changes in the peri-infarct region. The mechanism ofMEMRI contrast agent, developed in collaboration with Eagle VisionPharmaceutical Inc., SeeMore™ (EVP1001), is based on an activeingredient consisting of non-chelated Mn²⁺ (12%) with calcium gluconate(10%). The non-chelated property of Mn²⁺ enables safe and rapid uptakeby the Ca²⁺-channels of the viable cells, allowing highly effectiveintracellular targeting of viable cardiac cells with high level ofsafety and unique magnetic properties. The Ca²⁺ provides for safety,avoiding the cardiac depression that occurs with intravascular Mn²⁺alone and does not alter the fundamental distribution or enhancementproperties of Mn²⁺. This combination of Mn²⁺ with Ca²⁺ provides theunique intracellular enhancing features of Mn²⁺ (that otherwise requiresradioactive tracers) while avoiding cardiac suppression or otheruntoward effects.

The chemical property of Mn²⁺ allows the active ingredient to traversereadily into the extra-cellular and -vascular space; however, Mn²⁺ doesnot enhance these regions without the presence of viable cells. It isactively taken up by live, active cells and binds to intracellularcomponents such as the mitochondria to provide significant intracellularenhancement. If there are no live, active cells, there will be little tono enhancement by manganese, unlike other available magnetic imagingagents which are nonspecific, extracellular enhancing agents. For anexample, gadolinium (Gd) does not get taken up into the cells. Instead,the infarct or edematous regions have an abnormally high “extracellularfluid” space relative to normal tissue and, therefore, shownonspecific/extracellular enhancement. MEMRI does not enhance suchconditions because its enhancement relies on the specific, active uptakeinto live cells.

The published data on EVP have demonstrated high dose safety studiesaimed at eliciting signs of toxicity at target organs. The safety indexof 45 to 90 corresponded more favorably than the safety index of widelyused clinical imaging agents (10 to 20 for iodinated X-ray products and20 to 60 for gadolinium chelate MRI products). The human dose escalationstudy demonstrated safety, tolerance, pharmacokinetic (PK) profile, andmultiple clinical parameters, which demonstrated safety at all dosestested. Phase I and II clinical trials have been completed anddemonstrated no significant safety concerns. This specific, activeintracellular uptake is one of the key attributes that offers potentialbenefit of cellular MRI of the myocardium. The labeling efficiency ofMn²⁺ in single cell culture of human bone marrow stem cells and humanembryonic stem cells was studied and published.

Basic MEMRI parameters, including T₁ and T₂ values, were measured andvalidated in different concentration of Mn²⁺ (0.01-3.0 mM). The datawere analyzed to extract T₁ and T₂ values through nonlinear least-squarefits to the inversion recovery and spin-echo decay curves, respectively.The MEMRI viability signal was validated by transducing the cells withluciferase RG to detect BLI signal. MEMRI signal was modulated by theCa²⁺ channel activity using a calcium-channel antagonist (verapamil) andagonist ((s)-Bay K8644), demonstrating subsequent decreased andincreased MEMRI signal, respectively. These unique in vivo propertiesallow the infarcted to take up negligible amount, peri-infarct regionsreduced amount, and normal tissue greatest amount for MEMRI enhancement.These properties enable precise determination of the baseline cardiacinjury and subsequent restoration following stem cell therapy bydelineating the direct changes in the regional viability at a cellularlevel. An FDA IND approval was obtained by the PI and a pilot clinicaltrial of 6 ischemic cardiomyopathy patients has demonstrated cleardelineation of peri-infarct injury.

Dual contrast MEMRI-DEMRI. Transmural DEMRI-positive myocardial regionswould traditionally indicate non-viable scar. However, at-riskmyocardium that falls outside the infarct region and inside the PIR maycontain viable cells. The novel dual contrast MEMRI-DEMRI highlights theeffective of the Mn contrast agent, pointing to the viablecardiomyocytes within the PIR of transmural delayed enhancement. Thediscrepancy lies within the PIR that are positive for both MEMRI(viable) and DEMRI (non-viable) signal. The PIRs also display lower SNRby MEMRI than remote zones and lower SNR by DEMRI than core infarctzones, reflecting the heterogeneity of PIR with significant populationof viable cardiomyocytes with intact Ca²⁺-channel function (MEMRIpositive) in the PIR of the adjacent necrotic tissue (DEMRI positive).

Our histopathological analysis of these overlapping zones of MEMRI andDEMRI signal demonstrate morphologically and ultrastructurally intactbut injured cardiomyocytes within DEMRI-positive zone consistent withprevious publications regarding DEMRI overestimation of scar volume.Clinically, there is a critical need to detect truly viable myocardiumat various time points post-MI to assist decisions on revascularization.Despite early-invasive revascularization, poorer outcomes have beendemonstrated in certain subsets of patients, prompting recent changes inrevascularization guideline. Thus, the patients in the peri-MI periodmay greatly benefit from accurate imaging of at-risk, viable myocardiumthat could be jeopardized.

MEMRI-DEMRI approach addresses an unmet need to predict the overallpropensity of PIR to develop ventricular arrhythmia and/or remodeling.More importantly, the effects of cell, revascularization, and/or medicaltherapies on PIR can be delineated through the detection of progressivechanges in regional viability and tissue heterogeneity.

Peri-infarct region (PIR) in ischemic cardiomyopathy. Ischemiccardiomyopathy (ICM) is the primary etiology of advanced heart failure(HF), the leading diagnosis of hospital admissions in the US. Clinicalstudies have confirmed that the high morbidity and mortality of HF areassociated with ventricular arrhythmias and LV remodeling in theperi-infarct region (PIR). Indeed, patients with a history of acute MIand LV dysfunction have a 6 month mortality >10%, one third of which isattributed to sudden cardiac death. The critical role of tissueheterogeneity in the PIR, independent of actual infarct size, has beenrecognized as an important substrate to trigger these ventriculararrhythmias.

Preclinical studies have confirmed that the heterogeneous PIR containsthe critical isthmus for ventricular tachycardia and successful therapyrequires ablation of the isthmus. Studies have also demonstrated thatrevascularization of the ischemic PIR results in a lower incidence ofventricular arrhythmias and LV dilatation. While revascularization maymitigate ventricular arrhythmias acutely, the Multicenter UnSustainedTachycardia Trial (MUSTT) noted that following a reperfused MI, patientswere increasingly likely to have ventricular arrhythmias as theirinfarct matured. Therefore, an accurate tissue characterization of thePIR is critical in determining which patients will benefit fromrevascularization and/or medical therapy targeted at PIR. Precise tissuecharacterization of the PIR predicts future cardiovascular events whiletraditional measures including scar presence, LVEF, and LV volumes didnot demonstrate such significance.

EXAMPLE 2

Dual contrast enhanced cardiac MRI using manganese and gadolinium inpatients with severe ischemic cardiomyopathy detects the peri-infarctregion (PIR)

Delayed Enhanced MRI (DEMRI) with gadolinium (Gd) is used as goldstandard for diagnosis of myocardial infarction. However, thenon-specific property of Gd overestimates the infarct size. Conversely,manganese (Mn2+) enters only the live, active cardiomyocytes via L-typeCa2+ channels. From our earlier work in animal MI models,manganese-enhanced MRI (MEMRI) has demonstrated its utility inidentifying the viable, nonviable, and injured myocardium. We performedthe “first in human” dual-contrast MEMRI-DEMRI to assess the efficacy ofMEMRI-DEMRI to identify the periinfarct region (PIR) in patients withsevere ischemic cardiomyopathy (ICM).

Methods

5 ICM patients (Class I-Ill CHF) have been enrolled (5 male, mean age60±7 years). Cardiac MRI was performed using a 3.0T MRI scanner (Signa3T HDx, GE HealthCare, USA) with an 8 channel cardiac coil (3.0T HDCardiac Array, GE HealthCare, USA). LV functional images and DEMRI wereacquired on the first day of this study, and MEMRI was acquired on thefollowing week. (1) LV function: SSFP, flip angle (FA) 45, slicethickness (ST) 8.0 mm, matrix 224×224, FOV 35.0 cm; (2) DEMRI: FGRE-IR,TR 6.0, TE 2.8, TI 200-300, FA 15, ST 8.0 mm, matrix 224×192, FOV 35.0cm, 0.2 mmol/kg Gd (Magnevist, Bayer HealthCare, Germany); and (3)MEMRI: FGRE-IR, TR 6.0, TE 2.8, TI 600-700, FA 15, ST 8.0 mm, matrix224×192, FOV 35.0 cm, 1 mmol/kg Mn contrast agent, as described in U.S.Pat. No. 5,980,863, were performed. The infarct volumes were determinedas 3 standard deviations (SDs) above mean on DEMRI and 2 SDs below meanon MEMRI using a Cardiac MRI Software (CMR42, Circle CardiovascularImaging Inc., Canada).

Results

The average LVEF was 35±4%. The % enhanced DEMRI infarct volume(34±11%*) was significantly (*p<0.05) higher than the % defect MEMRIinfarct volume (14±3%). The PIR was calculated as the difference betweenDEMRI and MERMRI. The mean % PIR per total LV and the mean % PIR perDEMRI enhancement were 20±12% (FIGS. 1) and 56±16% (FIG. 2),respectively.

Conclusions

The non-viable myocardium volume, appearing as MEMRI defect, wassignificantly smaller than the DEMRI enhancement. The discrepancybetween DEMRI and MEMRI represents the PIR or “area-at-risk”. Therefore,our results show that the dual MEMRI-DEMRI contrast can clearlydelineate the PIR by integrating the biology of viable myocardium andanatomy of non-viable myocardium. This dual contrast approach delineatesthe area-at-risk and predicts clinical outcomes from revascularization.

We claim:
 1. A method for analysis of heterogeneous tissue, the methodcomprising performing manganese enhanced MRI on the tissue, anddetermining the presence of zone of tissue where intracellularaccumulation of Mn²⁺ generates high signal from the viable cells; nosignal from the non-viable cells; and intermediate signal from viablebut injured cells.
 2. The method of claim 1, wherein the heterogeneoustissue comprises a myocardial infarct and peri-infarct region.
 3. Themethod of claim 1, wherein the heterogenous tissue is atrial walltissue.
 4. The method of claim 1, wherein imaging is performed with acontrast agent comprising non-chelated Mn²⁺ and calcium gluconate. 5.The method of claim 1, wherein the analysis further comprises asequential or concomitant analysis by delayed-enhanced MRI (DEMRI) thatdistributes primarily within the extracellular space to generate abright MRI signal in infarcted tissue.
 6. The method of claim 5, whereinthe difference between the infarct size obtained with DEMRI issubtracted from the infarct size obtained with MEMRI to provide ameasurement of viable cells in the heterogeneous tissue.
 7. The methodof claim 1, wherein the analysis is performed for at least 2 timepoints.
 8. The method of claim 7, wherein therapeutic intervention isperformed between the two time points.
 9. The method of claim 8, whereinthe therapeutic intervention is performed in the context of a clinicaltrial.